1Universitätsklinikum Tübingen Innere Medizin III, Kardiologie und Angiologie Tübingen, Deutschland; 2Universitätsklinikum Tübingen Innere Medizin III, Kardiologie und Kreislauferkrankungen Tübingen, Deutschland
Background: Degenerative aortic stenosis (AS) is driven by progressive inflammatory and fibro-calcific processes regulated by infiltration of circulating peripheral monocytes and platelets that secrete pro-inflammatory mediators. Pro-inflammatory cytokines and chemokines play a critical role in the regulation of phenotypic changes of valvular interstitial cells (VIC) that result in VIC differentiation towards an inflammatory or osteogenic phenotype. Among those, macrophage migration inhibitory factor (MIF), a pro-inflammatory and pro-atherogenic regulator protein, which boosts vascular inflammation, might be a key trigger molecule for valve inflammation. The impact of platelet- and monocyte-derived MIF on VIC phenotypes mirroring local valvular inflammation and mineralization is presently unknown.
Methods: To address the functional consequences of MIF on VICs, we performed in vitro cell culture experiments. We analyzed the effect of recombinant MIF and the MIF antagonist ibudilast on inflammatory and osteogenic changes of VICs. In brief, human VICs plated in 12-well plates were cultured in inflammatory or osteogenic medium for seven days. VICs were incubated with either human recombinant MIF protein (R&D, MAB289-MF), the MIF antagonist ibudilast (Sigma-Aldrich I0157), or the combination of both over seven days before harvest and analysis by an intracellular staining wit anti-Vimentin AF647 (R&D, MAB210, anti-alpha smooth muscle actin (αSMA) AF488 (R&D, MAB1420), and anti-alkaline phosphatase (ALP) PE (R&D, MAB1448). Cells were acquired using LSR II Fortessa flow cytometer with the DIVA software and were further analyzed using FlowJo 10.6.2 software.
Results: We stimulated human VICs with recombinant MIF and the MIF antagonist ibudilast to evaluate morphologic alterations of VICs indicating inflammatory and myofibroblastic transformation compared to osteogenic phenotypes. The inflammatory phenotype of VICs was defined by the increased expression of vimentin and α-SMA. To mimic an either inflammatory or osteogenic driven environment we performed the cell culture experiments with pro-inflammatory medium and pro-osteogenic medium, respectively, to either influence a differentiation towards an inflammatory versus an osteogenic phenotype. We found that depending on the culture medium (inflammatory versus osteogenic) MIF enhances prominently an inflammatory phenotype of VICs determined by significantly increased vimentin and α-SMA expression, (p<0.0001), which could be attenuated by the MIF antagonist ibudilast (p<0.05). As osteogenic differentiation of VICs is characterized by increased activity and expression of alkaline phosphatase (ALP), we also analysed ALP expression in in vitro cell culture. Here, we detected that MIF stimulation also results in increased ALP expression in osteogenic-driven VICs compared to VICs in inflammatory medium (p<0.0001). Thus, we conclude that MIF is a major trigger for inflammatory changes of VICs and thereby for disease progression in aortic stenosis.
Conclusion: We report a completely novel role of MIF regarding the differentiation of VICs towards an inflammatory valvular phenotype, where MIF stimulation was identified as a key trigger component. We also demonstrate marked attenuation of the inflammatory VIC phenotype by the MIF-inhibitor ibudilast. Therapeutic targeting of MIF could serve as a novel strategy to limit progressive inflammation and finally calcification in aortic stenosis.